Genome Med; auth.: group Reymond

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles

Sissy Bassani 1 2Jacqueline Chrast 1Giovanna Ambrosini 3 4Norine Voisin 1 5Frédéric Schütz 6Alfredo Brusco 7 8Fabio Sirchia 7 8 9 10Lydia Turban 11Susanna Schubert 11Rami Abou Jamra 11Jan-Ulrich Schlump 12Desiree DeMille 13Pinar Bayrak-Toydemir 14Gary Rex Nelson 14Kristen Nicole Wong 14Laura Duncan 15 16Mackenzie Mosera 15Christian Gilissen 17Lisenka E L M Vissers 17Rolph Pfundt 17Rogier Kersseboom 18Hilde Yttervik 19Geir Åsmund Myge Hansen 19Marie Falkenberg Smeland 20Kameryn M Butler 21Michael J Lyons 21Claudia M B Carvalho 22 23Chaofan Zhang 23James R Lupski 23 24 25 26Lorraine Potocki 23 26Leticia Flores-Gallegos 27Rodrigo Morales-Toquero 27Florence Petit 28Binnaz Yalcin 29Annabelle Tuttle 30Houda Zghal Elloumi 30Lane McCormick 31Mary Kukolich 31Oliver Klaas 32Judit Horvath 32Marcello Scala 33 34Michele Iacomino 34Francesca Operto 35Federico Zara 33 34Karin Writzl 36 37Aleš Maver 36Maria K Haanpää 38Pia Pohjola 38Harri Arikka 39Anneke J A Kievit 40Camilla Calandrini 40Christian Iseli 3 4Nicolas Guex 3 4Alexandre Reymond 41

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Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects.

Methods: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants.

Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation.

Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.